(i) Field of the Invention:
The present invention relates to a peptide of linear, non-cyclic structure consisting of or comprising 17 to 23 amino acids, wherein the amino acids in positions 1 to 23, counted from the N-terminus, are as follows (1) G, S or lacking; (2) C or lacking; (3) K or R; (4) K or R; (5) Y, W or F; (6) K or R; (7) K or R; (8) F, W or L; (9) K or R; (10) K or L or lacking; (11) W, L or F; (12) K or R; (13) F, Y or C; (14) K or R; (15) G or Q; (16) K or R; (17) F, L or W; (18) F or W; (19) F, L or W; (20) W or F; (21) C or lacking; (22) F or G or lacking (23) G or lacking. Further, the invention relates to a nucleic acid molecule encoding the peptide of the invention, an expression vector comprising the nucleic acid molecule of the invention, a host cell which may be grown in cell culture comprising the vector of the invention, and a method of producing the peptide of the invention comprising culturing the host cell of the invention and collecting the peptide produced. Also, the present invention relates to a pharmaceutical composition comprising the peptide of the invention, the peptide produced by the method of the invention, the nucleic acid molecule of the invention, the expression vector of the invention or the host cell of the invention and a kit comprising the peptide of the invention, the peptide produced by the method of the invention, the nucleic acid molecule of the invention, the expression vector of the invention or the host cell of the invention.
In this specification, a number of documents including patent applications and manufacturer's manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
(ii) Description of the Related Art:
In Germany alone more than 60000 people per year die from bacterial blood poisoning (sepsis). A new approach to fight this and other conditions has been the use of synthetic antimicrobial peptides (AMPs) on the basis of particular motifs of human and other proteins, which bind to bacterial pathogenicity factors such as lipopolysaccharide (LPS). It is known that the latter molecules are to a considerable degree responsible for inflammation and infection.
The approach to use AMPs has gained some interests in the last years. Usually, AMPs are based on LPS-binding defense proteins such as lactoferrin, granulysins and cationic antimicrobial peptides (CAP) (Andersson et al., 1996, Garidel et al., 2007, Ramamoorthy et al., 2006, Vallespi et al., 2003). Most of these studies, however, concentrated on killing the infectious bacteria rather than neutralizing free LPS. The existence of isolated LPS, released from bacteria by the action of the immune system or simply by cell dividing, is one of the main problems in the anti-septic fight.
Some studies have used the LPS-binding domain of the LALF-protein (Hoess et al., 1993, Paus et al., 2002), and have synthesized linear and cyclic peptides, with which it was possible to obtain a certain LPS-neutralization in vitro, but also in vivo, e.g. in a mouse model of endotoxicity (Dankesreiter et al., 2000, Garidel et al., 2007, Hoess et al., 1993, Leslie et al., 2006, More et al., 2006, Ried et al., 1996, Vallespi et al., 2003). It was found that some of these peptides have a sufficient half life in serum. However, the question of whether these compounds suppress the endotoxicity effectively and are suitable as possible anti-sepsis agents remained unanswered.
Comprehensive biophysical studies were performed, in which the essential parameters important for LPS-neutralization by particular cyclic AMPs or peptides based on porcine NK-lysin or human granulysin could be characterized (Andrä et al., 2004, Andrä et al., 2007, Andrä et al., 2004, Andrä et al., 2004, Andrä et al., 2007, Chen et al., 2007). Among these parameters are surface potential of the LPS head group, the fluidity of the lipid A acyl chains, the lipid A aggregate structure, and the incorporation into phospholipid liposomes in the absence and the presence of the LPS-binding protein LBP.
It was found that the LPS-neutralization by these AMPs was not sufficiently high to use them in animal experiments. The problem to obtain suitable AMP compounds is a severe problem due to the extremely high number of combination possible starting from the 20 proteinogenic amino acids. An approach to develop suitable AMP must include their ability to act antimicrobially as well as to neutralize LPS. For this, a detailed knowledge of the size, conformation, and aggregate structure of LPS/lipid A is necessary. Some of these parameters have been determined in previous reports (Brandenburg, 1993, Brandenburg et al., 1999, Brandenburg et al., 1990, Brandenburg et al., 1992, Brandenburg et al, 1997, Brandenburg et al., 2000, Brandenburg et al., 2002, Brandenburg et al., 1993, Brandenburg et al., 1998, Brandenburg et al., 1996), for a review see Brandenburg and Wiese, 2004.
However, even with this information available, there is still a need of providing peptides with excellent antimicrobial properties.